Cromolyn derivatives and related methods of imaging and treatment

ABSTRACT

Novel cromolyn analogs useful as imaging agents for detecting atherosclerotic plaques and for treating atherosclerosis and Alzheimer&#39;s Disease, and methods of making the cromolyn analogs, are disclosed. The cromolyn analogs have the general formula: 
                         
wherein: X is OH, C 1 -C 6  alkoxyl; Y and Z are independently selected form a C 1 -C 6  alkyl, C 1 -C 6  alkoxyl, halogen, un-substituted or C 1 -C 6  substituted amine,  18 F,  19 F, or H; and n is 1, 2, or 3; and wherein for structure (I), if n are both 1 and Y and Z are both H and X is OH.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 14/059,924, filed Oct. 22, 2013, which is a continuation-in-partapplication of U.S. application Ser. No. 13/146,842 filed Nov. 16, 2011,now U.S. Pat. No. 8,617,517, issued on Aug. 5, 2010, which representsthe U.S. National Stage of PCT/US2010/22495, filed Jan. 29, 2010, whichclaims benefit of U.S. Provisional application 61/148,245, filed Jan.29, 2009, the contents of each of which are expressly incorporated byreference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

FIELD OF THE INVENTION

The invention relates generally to medical imaging and diseasetreatment. In particular, the invention is directed to cromolynderivatives for use in positron emission tomography (PET) imaging andrelated methods of diagnosis and therapeutic treatment.

BACKGROUND OF THE INVENTION

Coronary artery disease is the leading cause of morbidity and mortalityin the United States and in most developed countries. Atherosclerosisand its complications such as myocardial infarction and stroke, ismainly responsible for coronary artery disease. All together,atherosclerosis accounts for at least forty-three percent of all deathin the United States affecting over 60 million people (American HeartAssociation, 2004).

Advances in basic science indicate coronary artery disease is aninflammatory process, characterized by a long cycle of irritation,injury, healing and re-injury to artery endothelial cells. There isgrowing evidence that mast cells are found in the various stages ofatherosclerosis, coronary inflammation and cardiac ischemia (Libby 2002;Fernex, 1968; Mor and Mekori, 2001; Kelly, Chi, et al., 2000; Sun,Sukhova, et al., 2007; Huang, Pang, et al., 2002). Mast cells, locatedin connective tissue, play an important role in helping the immunesystem defend tissues from disease by activating the release ofintracellular mediators (degranulation), as well as attracting other keyplayers of the immune defense system to areas of the body where they areneeded. In response to vascular injury, cardiac mast cells interact withlipoproteins to deliver lipids to macrophages, and to release a largevariety of cytokines that affect smooth muscle cells and T lymphocytes.This process can develop into the more advanced and complex occlusivelesions, termed fibrous plaques. Other pro-inflammatory mediatorsreleased by mast cells are histamine, which can constrict thecoronaries, and cytokines IL-6 and IFN-gamma, which induce degradationof the extracellular matrix and the death of smooth muscle cells in thewall of the aorta, weakening the walls and allowing it to dilate. Thus,the inflammatory response stimulates endothelial dysfunction causingmigration and proliferation of smooth muscle cells that becomeintermixed in the area of inflammation to form fibrous plaques andcomplicated lesions.

Disodium cromoglicate, termed “cromolyn,” is the disodium salt ofcromoglicic acid. It is used as an anti-inflammatory medication.Cromolyn is described in the literature as a mast cell stabilizer sinceit works by preventing the release of mediators such as the vasoactiveand pro-arrhythmogenic chemical histamine and cytokines from mast cellsthus stabilizing inflammatory cells. Prevention of mediator release isthought to result from indirect blockade of the entry of calcium ionsinto the membrane of sensitized mast cells. Cromolyn has also been shownto inhibit the movement of other inflammatory cells such as neutrophils,eosinophils, and monocytes (8).

Recent studies in mice have demonstrated that systemic mast cellactivation during atherogenesis leads to plaque formation (Bot, deJager, et al., 2007). Furthermore, treatment of the animals with themast cell stabilizer cromolyn prevented dinitrophenyl-albumin-inducedplaque expansion. In another study, cardiac mast cell activation wasstudied in mice after stress-related coronary inflammation (Huang, Pang,et al., 2003). Activated mast cells were found adjacent toatherosclerotic vessels. In cromolyn treated mice, release of thepro-inflammatory cytokine interleukin-6 (IL-6) present in mast cells,was partially inhibited.

There is growing evidence that activated cardiac mast cells areincreased in association with coronary inflammation, myocardialinfarction, as well as ischemic cardiomyopathy. Moreover, mast cells canpromote the formation of human atherosclerotic lesions by causingendothelial dysfunction of the heart's arteries that lead to plaquebuildup. Since cromolyn targets sensitize mast cells, a labeled cromolynanalog potentially could serve as a diagnostic probe for early detectionof coronary artery disease.

As can be appreciated, it would be desirable to obtain new imagingagents useful for detecting degenerative diseases in human subjects. Inparticular, novel imaging probes that associate with markers ofinflammation such as mast cells would facilitate the early detection ofinflammatory diseases such as atherosclerosis by utilizing sensitive andnon-invasive approaches such as PET or MRI imaging. Such new compoundsmay also provide unexpected therapeutic benefits for treatment ofconditions including, but not limited to, inflammation, infection,atherosclerosis and Alzheimer's Disease.

SUMMARY OF THE INVENTION

The inventors show herein the synthesis and use of new cromolynderivatives. Accordingly, the invention provides imaging agents suitablefor imaging sites of inflammatory activity, including atheroscleroticplaques in the heart, brain and carotid artery, and β-amyloid plaques inthe brain. In addition, the invention provides compounds that providetherapeutic effects in the treatment of various conditions including,but not limited to, inflammation, infection, atherosclerotic plaque, andAlzheimer's Disease.

In a first aspect, the invention provides a compound having the formula:

or an ester or salt of (I) or (II); wherein: X is OH, C₁-C₆ alkoxyl, Yand Z are independently selected from a C₁-C₆ alkyl, C₁-C₆ alkoxyl,halogen, un-substituted or C₁-C₆ substituted amine, ¹⁸F, ¹⁹F, or H; andn is 1, 2, or 3; and wherein for structure (I), if n are both 1 and Yand Z are both H, X is OH.

In certain embodiments, X is ¹⁸F or ¹⁹F and, more preferably, Y and Zare hydrogen. A particularly preferred compound has the structure:

or is a salt or ester thereof.

In another embodiment, at least one of Y and Z is ¹⁸F or ¹⁹F and, morepreferably, X is OH. Particularly preferred compounds have thestructures:

or are corresponding salts or esters thereof.

In an alternative embodiment, the compound lacks a radiolabel and,preferably, X is OH and Y and Z are hydrogen, except that in such anembodiment comprising structure (I) wherein X is OH and Y and Z arehydrogen, both n cannot be 1.

Preferred compounds of the invention, particularly for imaging purposes,localize to atherosclerotic plaques in the heart, brain and/or carotidartery of a subject, or to 3-amyloid plaques in the brain of a subject.

In another aspect, the compounds of the invention are provided in theform of a pharmaceutically appropriate dosage of one or more of thecompounds described and claimed herein formulated with apharmaceutically acceptable carrier.

As can be appreciated, the compounds of the invention are useful forimaging in other modalities in addition to PET imaging. Exemplarycompounds may be optionally isotopically labeled with isotopes such asthe ¹⁹F isotope, or ¹³C isotope to facilitate nuclear magnetic resonanceimaging (MRI).

In yet another aspect of the invention, a method for providing apositron emission tomography (PET) scan of a subject is provided. Such amethod includes steps of: (a) administering to a subject a compoundcontaining an ¹⁸F label as described and claimed herein; and (b) imaginggamma rays emitted due to the compound within the subject in order toprovide a PET scan of the compound contained in the subject.

In preferred methods, the presence, absence or level of the compoundwithin the subject is indicative of a disease state including, but notlimited to, atherosclerotic plaque alternatively present in the heart,brain, or carotid artery of the subject.

The subject is preferably a living animal, most preferably a human.

The compound is typically administered to the subject via intravenous(IV) injection.

In certain alternative methods, an additional step of contrast imagingthe subject by magnetic resonance imaging (MRI) or x-ray computedtomography (CT) is included.

In yet another embodiment, the invention provides a method for providinga magnetic resonance image of a subject. Such a method includes stepsof: (a) administering to a subject a compound containing an ¹⁸F labelaccording to the present invention; and (b) imaging the subject in orderto obtain a magnetic resonance image of the compound contained withinthe subject.

The presence, absence or level of the compound within the subject isindicative of a disease state, preferably atherosclerotic plaque presentin the heart, brain, or carotid artery of the subject.

The invention further encompasses treatment methods, including treatmentof atherosclerotic plaque in a subject. Such a method includes steps ofadministering to a subject an effective dosage of a compound of theinvention, whereby the atherosclerotic plaque is treated in the subject.

In an alternative method, the invention provides a method of treatingAlzheimer's Disease in a subject including the steps of administering toa subject an effective dosage of an inventive compound, wherebyAlzheimer's Disease is treated in the subject.

Also provided by the invention are novel methods of efficientlypreparing fluorinated compounds. Such methods provide for quickersynthesis and purification than is seen in conventional methods.Accordingly, the methods are particularly suited for fluorinatingcompounds with radiolabeled fluorine for use in imaging applications.

In some embodiments of a method of preparation, a fluoride moiety iscontacted with an organic compound having a triflate or tosylate moietyon an aliphatic carbon atom under anhydrous or aprotic conditions. Undersuch conditions, the fluoride moiety acts as a nucleophile and thetriflate or tosylate acts as a leaving group in a nucleophilicsubstitution reaction, resulting in the fluorination of the organiccompound. Preferably, the fluoride moiety used in the method is F-18.

In certain such embodiments, the organic compound contacted with thefluoride moiety is1,3-bis[(tolylsulfonyl)oxy]-2-[(trifluoromethyl)sulfonyl]oxy-propane,and the resulting product is further reacted with other compounds toprovide a fluorinated cromolyn derivative. In one embodiment, cromolynderivatives are provided wherein X in structure (I) or structure (II)above is a fluorine atom. Preferably, the fluorine atom is radiolabeledF-18.

In yet other embodiments of a method of preparation, a fluoride moietyis contacted under anhydrous or aprotic conditions with an organiccompound having an activated aromatic ring wherein the aromatic ring islinked to a nitro group, a substituted ammonium ion, a substitutedsulphonium ion, a substituted phosphonium ion, or a halogen. Under suchconditions, the fluoride moiety exchanges for the nitro group,substituted ammonium ion, substituted sulphonium ion, substitutedphosphonium ion, or halogen, resulting in the fluorination of theorganic compound on the aromatic ring. Preferably, the fluoride moietyused in the method is F-18.

In certain such embodiments, the organic compound contacted with thefluoride moiety is a cromolyn derivative-based substituted ammonium saltwherein the nitrogen atom of the substituted ammonium ion is attached toan aromatic ring of the chromolyn derivative. In some such embodiments,cromolyn derivatives are provided wherein Y or Z in structure (I) orstructure (II) above is a fluorine atom. Preferably, the fluorine atomis radiolabeled F-18.

Of course, the invention also contemplates the use of a compound asdescribed and claimed herein for the manufacture of an injectable dosagefor the in vivo imaging of a subject as well as a medicament for thetreatment of disease conditions such as atherosclerotic plaque andAlzheimer's Disease. In addition, the invention encompasses the use ofthe present compounds in in vivo imaging of a subject and treatment ofdisease conditions.

In one embodiment, the present invention is a method for treatingAlzheimer's Disease in a subject comprising the step of administering bysystemic delivery an effective amount of a compound having the formula:

or a salt or ester of (I) or (II); wherein: X is OH, C₁-C₆ alkoxyl, Yand Z are independently selected from a C₁-C₆ alkyl, C₁-C₆ alkoxyl,halogen, un-substituted or C₁-C₆ substituted amine, ¹⁸F, ¹⁹F, or H; andn is 1, 2, or 3, wherein Alzheimer's Disease is treated in the subject.

In another embodiment, the present invention is a method for inhibitingthe polymerization of amyloid-beta peptide oligomers in a subjectcomprising the step of administering by systemic delivery an effectiveamount of a compound having the formula:

or a salt or ester of (I) or (II); wherein: X is OH, Y and Z areindependently selected from a C₁-C₆ alkyl, C₁-C₆ alkoxyl, halogen,un-substituted or C₁-C₆ substituted amine, ¹⁸F, ¹⁹F, or H; and n is 1,2, or 3, wherein the polymerization of amyloid-beta peptide oligomers inthe subject is inhibited.

Other objects, features and advantages of the present invention willbecome apparent after review of the specification, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the water maze recorded data of in vivo cromolyn andibuprofen treatment of transgenic mice-modeling like Alzheimer'sDisease. The results indicate that treated transgenic mice have closelybehavior to wild type normal control group.

FIG. 2A and FIG. 2B illustrate the measurement of TBS soluble Aβ levelby WAKO ELISA. The experiments show that TBS Aβ level decreasesfollowing by treatment of cromolyn sodium with dose-dependency. FIG. 2Ashows that Aβ-40 level decreases following by treatment of cromolynsodium with dose-dependency. FIG. 2B shows that Aβ-42 level decreasesfollowing by treatment of cromolyn sodium with dose-dependency. N=3 or 5animals per group, average±SE. The p value is significant using one-wayANOVA test (Bonferroni's test). Both of total soluble Aβ (as shown asGdn+) and monomeric Aβ (as shown as Gdn−) decease after the addition ofcromolyn sodium. The dose of 2.1 mg/kg of cromolyn sodium was enough todecrease TBS soluble Aβ.

FIG. 3A, FIG. 3B, and FIG. 3C illustrate the measurement of TBS solubleAβ oligomer level IBL oligomer ELISA. The experiments show that Aβoligomer level was not changed following the treatment with cromolynsodium. FIG. 3A shows the experiments of IBL Aβ oligomer ELISA(82E1-82E1). FIGS. 3B and 3C show the difference the experiments withGdn and those without Gdn using Aβ WAKO ELISA. N=3 or 5 animals pergroup, average±SE. The p value is not significant using one-way ANOVAtest (Bonferroni's test). Both ELISA (IBL oligomer ELISA and thedifferences between with and without Gdn using WAKO ELISA) showed thatoligomer level was not changed following the treatment with cromolynsodium.

FIG. 4 illustrates the biodistribution of cromolyn Compound A followingintravenous injection in mice. In FIG. 5, a 5, 30 or 60 minute,corresponding to Series 1, 2 or 3, respectively in the graph, brainuptake shows 1% accumulation with little or no washout for the periodmeasured.

FIG. 5 illustrates Aβ aggregation test in the absence of cromolyn. Theexperiment was assayed by thioflavin fluorescent intensity kinetics.

FIG. 6 illustrate Aβ aggregation test after the addition of cromolyn(CO399) or its ¹⁹F derivative (TS734). The addition of cromolyn (CO399)and its ¹⁹F derivative (TS734) at nanomolar concentration showsinhibition of Aβ aggregation.

FIG. 7 illustrates the side view of the relative structures andlocations of cromolyn and Aβ after cromolyn binds Aβ through a bindingmodel simulation.

FIG. 8 illustrate the top view of the relative structures and locationsof cromolyn and Aβ after cromolyn binds Aβ through a binding modelsimulation.

DETAILED DESCRIPTION OF THE INVENTION I. In General

Before the present materials and methods are described, it is understoodthat this invention is not limited to the particular methodology,protocols, materials, and reagents described, as these may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby any later-filed nonprovisional applications.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. As well, the terms “a” (or “an”),“one or more” and “at least one” can be used interchangeably herein. Theterms “comprising” and variations thereof do not have a limiting meaningwhere these terms appear in the description and claims. Accordingly, theterms “comprising”, “including”, and “having” can be usedinterchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications and patentsspecifically mentioned herein are incorporated by reference for allpurposes including describing and disclosing the chemicals, instruments,statistical analysis and methodologies which are reported in thepublications which might be used in connection with the invention. Allreferences cited in this specification are to be taken as indicative ofthe level of skill in the art. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

The terminology as set forth herein is for description of theembodiments only and should not be construed as limiting of theinvention as a whole. Unless otherwise specified, “a,” “an,” “the,” and“at least one” are used interchangeably and mean one or more than one.

As used herein, the term “organic group” is used for the purpose of thisinvention to mean a hydrocarbon group that is classified as an aliphaticgroup, cyclic group, or combination of aliphatic and cyclic groups(e.g., alkaryl and aralkyl groups). In the context of the presentinvention, suitable organic groups for cromolyn derivatives of thisinvention are those that do not interfere with the cromolyn derivativesimaging activity. In the context of the present invention, the term“aliphatic group” means a saturated or unsaturated linear or branchedhydrocarbon group. This term is used to encompass alkyl, alkenyl, andalkynyl groups, for example.

The terms “hydroxy” and “hydroxyl” refer to the group —OH.

The term “oxo” refers to the group ═O.

The term “carboxylate” or “carboxyl” refers to the group —COO— or —COOH.

The term “cyano” refers to the group —CN.

The term “nitro” refers to the group —NO₂.

The term “amino” refers to the group —NH₂.

The term “acyl” or “aldehyde” refers to the group —C(═O)H.

The term “amido” or “amide” refers to the group —C(O)NH₂.

The term “aminoacyl” or “acylamino” refers to the group —NHC(O)H.

The term “thiol” refers to the group —SH.

The term “thioxo” refers to the group ═S.

The term “sulfinyl” refers to the group —S(═O)H.

The term “sulfonyl” refers to the group —SO₂H.

The term “sulfonylamido” or “sulfonamide” refers to the group —SO₂NH₂.

The term “sulfonate” refers to the group SO₃H and includes groups havingthe hydrogen replaced with, for example a C₁₋₆alkyl group(“alkylsulfonate”), an aryl (“arylsulfonate”), an aralkyl(“aralkylsulfonate”) and so on. C₁₋₃sulfonates are preferred, such asfor example, SO₃Me, SO₃Et and SO₃Pr.

The term “isomers”, as used herein, refer to stereoisomers,diastereomers, enantiomers and tautomers. “Tautomers” may be isomersthat are readily interconvertable by rapid equilibrium. For example,carbonyl compounds that have a hydrogen on their alpha-carbon arerapidly interconverted with their corresponding enols.

As used herein, the terms “alkyl”, “alkenyl”, and the prefix “alk-” areinclusive of straight chain groups and branched chain groups and cyclicgroups, e.g., cycloalkyl and cycloalkenyl. Unless otherwise specified,these groups contain from 1 to 20 carbon atoms, with alkenyl groupscontaining from 2 to 20 carbon atoms. In some embodiments, these groupshave a total of at most 10 carbon atoms, at most 8 carbon atoms, at most6 carbon atoms, or at most 4 carbon atoms. Cyclic groups can bemonocyclic or polycyclic and preferably have from 3 to 10 ring carbonatoms. Exemplary cyclic groups include cyclopropyl, cyclopropylmethyl,cyclopentyl, cyclohexyl, adamantyl, and substituted and unsubstitutedbornyl, norbornyl, and norbornenyl.

The term “heterocyclic” includes cycloalkyl or cycloalkenyl non-aromaticrings or ring systems that contain at least one ring heteroatom (e.g.,O, S, N).

Unless otherwise specified, “alkylene” and “alkenylene” are the divalentforms of the “alkyl” and “alkenyl” groups defined above. The terms,“alkylenyl” and “alkenylenyl” are used when “alkylene” and “alkenylene”,respectively, are substituted. For example, an arylalkylenyl groupcomprises an alkylene moiety to which an aryl group is attached.

The term “haloalkyl” is inclusive of groups that are substituted by oneor more halogen atoms, including perfluorinated groups. This is alsotrue of other groups that include the prefix “halo-”. Examples ofsuitable haloalkyl groups are difluoromethyl, trifluoromethyl, and thelike. “Halogens” are elements including chlorine, bromine, fluorine, andiodine.

The term “aryl” as used herein includes monocyclic or polycyclicaromatic hydrocarbons or ring systems. Examples of aryl groups includephenyl, naphthyl, biphenyl, fluorenyl and indenyl. Aryl groups may besubstituted or unsubstituted. Aryl groups include aromatic annulenes,fused aryl groups, and heteroaryl groups. Aryl groups are also referredto herein as aryl rings.

Unless otherwise indicated, the term “heteroatom” refers to the atoms O,S, or N.

The term “heteroaryl” includes aromatic rings or ring systems thatcontain at least one ring heteroatom (e.g., O, S, N). In someembodiments, the term “heteroaryl” includes a ring or ring system thatcontains 2 to 12 carbon atoms, 1 to 3 rings, 1 to 4 heteroatoms, and O,S, and/or N as the heteroatoms. Suitable heteroaryl groups includefuryl, thienyl, pyridyl, quinolinyl, isoquinolinyl, indolyl, isoindolyl,triazolyl, pyrrolyl, tetrazolyl, imidazolyl, pyrazolyl, oxazolyl,thiazolyl, benzofuranyl, benzothiophenyl, carbazolyl, benzoxazolyl,pyrimidinyl, benzimidazolyl, quinoxalinyl, benzothiazolyl,naphthyridinyl, isoxazolyl, isothiazolyl, purinyl, quinazolinyl,pyrazinyl, 1-oxidopyridyl, pyridazinyl, triazinyl, tetrazinyl,oxadiazolyl, thiadiazolyl, and so on.

The terms “arylene” and “heteroarylene” are the divalent forms of the“aryl” and “heteroaryl” groups defined above. The terms “arylenyl” and“heteroarylenyl” are used when “arylene” and “heteroarylene”,respectively, are substituted. For example, an alkylarylenyl groupcomprises an arylene moiety to which an alkyl group is attached.

The term “fused aryl ring” includes fused carbocyclic aromatic rings orring systems. Examples of fused aryl rings include benzo, naphtho,fluoreno, and indeno.

The term “annulene” refers to aryl groups that are completely conjugatedmonocyclic hydrocarbons. Examples of annulenes include cyclobutadiene,benzene, and cyclooctatetraene. Annulenes present in an aryl group willtypically have one or more hydrogen atoms substituted with other atomssuch as carbon.

When a group is present more than once in any formula or schemedescribed herein, each group (or substituent) is independently selected,whether explicitly stated or not. For example, for the formula —C(O)NR₂each of the two R groups is independently selected.

As a means of simplifying the discussion and the recitation of certainterminology used throughout this application, the terms “group” and“moiety” are used to differentiate between chemical species that allowfor substitution or that may be substituted and those that, in theparticular embodiment of the invention, do not so allow for substitutionor may not be so substituted. Thus, when the term “group” is used todescribe a chemical substituent, the described chemical materialincludes the unsubstituted group and that group with nonperoxidic O, N,S, Si, or F atoms, for example, in the chain as well as carbonyl groupsor other conventional substituents. Where the term “moiety” is used todescribe a chemical compound or substituent, only an unsubstitutedchemical material is intended to be included. For example, the phrase“alkyl group” is intended to include not only pure open chain saturatedhydrocarbon alkyl substituents, such as methyl, ethyl, propyl,tert-butyl, and the like, but also alkyl substituents bearing furthersubstituents known in the art, such as hydroxy, alkoxy, alkylsulfonyl,halogen atoms, cyano, nitro, amino, carboxyl, etc. Thus, “alkyl group”includes ether groups, haloalkyls, nitroalkyls, carboxyalkyls,hydroxyalkyls, sulfoalkyls, etc. On the other hand, the phrase “alkylmoiety” is limited to the inclusion of only pure open chain saturatedhydrocarbon alkyl substituents, such as methyl, ethyl, propyl,tert-butyl, and the like.

The invention is inclusive of the compounds described herein (includingintermediates) in any of their pharmaceutically acceptable forms,including isomers (e.g., diastereomers and enantiomers), tautomers,salts, solvates, polymorphs, prodrugs, and the like. In particular, if acompound is optically active, the invention specifically includes eachof the compound's enantiomers as well as racemic mixtures of theenantiomers. It should be understood that the term “compound” includesany or all of such forms, whether explicitly stated or not (although attimes, “salts” are explicitly stated).

“Pharmaceutically acceptable” as used herein means that the compound orcomposition or carrier is suitable for administration to a subject toachieve the treatments described herein, without unduly deleterious sideeffects in light of the necessity of the treatment.

The term “therapeutically effective amount” or “pharmaceuticallyappropriate dosage”, as used herein, refers to the amount of thecompounds or dosages that will elicit the biological or medical responseof a subject, tissue or cell that is being sought by the researcher,veterinarian, medical doctor or other clinician.

As used herein, “pharmaceutically-acceptable carrier” includes any andall dry powder, solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic agents, absorption delaying agents, and thelike. Pharmaceutically-acceptable carriers are materials, useful for thepurpose of administering the compounds in the method of the presentinvention, which are preferably non-toxic, and may be solid, liquid, orgaseous materials, which are otherwise inert and pharmaceuticallyacceptable, and are compatible with the compounds of the presentinvention. Examples of such carriers include, various lactose, mannitol,oils such as com oil, buffers such as PBS, saline, polyethylene glycol,glycerin, polypropylene glycol, dimethylsulfoxide, an amide such asdimethylacetamide, a protein such as albumin, and a detergent such asTween 80, mono- and oligopolysaccharides such as glucose, lactose,cyclodextrins and starch.

The term “administering” or “administration”, as used herein, refers toproviding the compound or pharmaceutical composition of the invention toa subject suffering from or at risk of the diseases or conditions to betreated or prevented.

The term “systemic delivery”, as used herein, refers to any suitableadministration methods which may delivery the compounds in the presentinvention systemically. In one embodiment, systemic delivery may beselected from the group consisting of oral, parenteral, intranasal,inhaler, sublingual, rectal, and transdermal administrations.

A route of administration in pharmacology and toxicology is the path bywhich a drug, fluid, poison, or other substance is taken into the body.Routes of administration may be generally classified by the location atwhich the substance is applied. Common examples may include oral andintravenous administration. Routes can also be classified based on wherethe target of action is. Action may be topical (local), enteral(system-wide effect, but delivered through the gastrointestinal tract),or parenteral (systemic action, but delivered by routes other than theGI tract), via lung by inhalation.

A topical administration emphasizes local effect, and substance isapplied directly where its action is desired. Sometimes, however, theterm topical may be defined as applied to a localized area of the bodyor to the surface of a body part, without necessarily involving targeteffect of the substance, making the classification rather a variant ofthe classification based on application location. In an enteraladministration, the desired effect is systemic (non-local), substance isgiven via the digestive tract. In a parenteral administration, thedesired effect is systemic, and substance is given by routes other thanthe digestive tract.

The examples for topical administrations may include epicutaneous(application onto the skin), e.g., allergy testing or typical localanesthesia, inhalational, e.g. asthma medications, enema, e.g., contrastmedia for imaging of the bowel, eye drops (onto the conjunctiva), e.g.,antibiotics for conjunctivitis, ear drops, such as antibiotics andcorticosteroids for otitis externa, and those through mucous membranesin the body.

Enteral administration may be administration that involves any part ofthe gastrointestinal tract and has systemic effects. The examples mayinclude those by mouth (orally), many drugs as tablets, capsules, ordrops, those by gastric feeding tube, duodenal feeding tube, orgastrostomy, many drugs and enteral nutrition, and those rectally,various drugs in suppository.

The examples for parenteral administrations may include intravenous(into a vein), e.g. many drugs, total parenteral nutritionintra-arterial (into an artery), e.g., vasodilator drugs in thetreatment of vasospasm and thrombolytic drugs for treatment of embolism,intraosseous infusion (into the bone marrow), intra-muscular,intracerebral (into the brain parenchyma), intracerebroventricular (intocerebral ventricular system), intrathecal (an injection into the spinalcanal), and subcutaneous (under the skin). Among them, intraosseousinfusion is, in effect, an indirect intravenous access because the bonemarrow drains directly into the venous system. Intraosseous infusion maybe occasionally used for drugs and fluids in emergency medicine andpediatrics when intravenous access is difficult.

Any route of administration may be suitable for the present invention.In one embodiment, the compound of the present invention may beadministered to the subject via intravenous injection. In anotherembodiment, the compounds of the present invention may be administeredto the subject via any other suitable systemic deliveries, such as oral,parenteral, intranasal, sublingual, rectal, or transdermaladministrations.

In another embodiment, the compounds of the present invention may beadministered to the subject via nasal systems or mouth through, e.g.,inhalation.

In another embodiment, the compounds of the present invention may beadministered to the subject via intraperitoneal injection or IPinjection.

As used herein, the term “intraperitoneal injection” or “IP injection”refers to the injection of a substance into the peritoneum (bodycavity). IP injection is more often applied to animals than to humans.In general, IP injection may be preferred when large amounts of bloodreplacement fluids are needed, or when low blood pressure or otherproblems prevent the use of a suitable blood vessel for intravenousinjection.

In animals, IP injection is used predominantly in veterinary medicineand animal testing for the administration of systemic drugs and fluidsdue to the ease of administration compared with other parenteralmethods.

In humans, the method of IP injection is widely used to administerchemotherapy drugs to treat some cancers, in particular ovarian cancer.Although controversial, this specific use has been recommended as astandard of care.

II. The Invention

In certain aspects, the invention is directed to radiolabeled cromolynanalogs for medical imaging of inflammatory sites such asatherosclerotic plaques in the heart, brain, or carotid artery of asubject. In other aspects, the present compounds, in radiolabeled orunlabeled form, are treatment agents for various disease conditionsincluding, e.g., atherosclerotic plaques and Alzheimer's Disease.

Disodium cromoglicate, or sometimes called cromolyn, is ananti-inflammatory medication. Cromolyn is understood to be a mast cellstabilizer and apparently works by preventing the release of mediatorssuch as the vasoactive and proarrhythmogenic chemical histamine andcytokines from mast cells thus stabilizing inflammatory cells.Prevention of mediator release is thought to result from indirectblockade of the entry of calcium ions into the membrane of sensitizedmast cells. Cromolyn has also been shown to inhibit the movement ofother inflammatory cells such as neutrophils, eosinophils, andmonocytes. The present inventors describe herein the manufacture and useof new analogs and new radiolabeled analogs of cromolyn for use aspotential agents for treatment, imaging and as biomarkers for followingprogression, treatment efficacy and prevention of atherosclerosis andβ-amyloid plaque formation. In certain embodiments, cromolyn analogs areradiolabeled with nuclides that allow PET and MRI imaging. The inventionfurther provides methods for the preparation and use of the compoundsfor targeting and treating active infection and other inflammatoryprocesses, such as atherosclerosis or, alternatively, Alzheimer'sDisease.

As can be appreciated, the compounds of the present invention may beused for several purposes. For instance, the described compounds are apotential research tool for animal studies; a diagnosis agent forclinicians; a biomarker for biology studies; a potential class of drugsto treat atherosclerosis or Alzheimer's Disease; a MRI imaging probe foratherosclerosis or Alzheimer's (e.g., a compound containing at least onefluorine atom which is an ¹⁹F isotope); and a PET probe foratherosclerosis or Alzheimer's diagnosis (e.g., a compound containing atleast one fluorine atom which is an ¹⁸F isotope or, alternatively, atleast one carbon atom which is a ¹³C isotope).

Cromolyn derivatives are expected to be beneficial for use in theimaging methods of the invention. As used herein, the term “cromolynderivative” is used interchangeably with the term “cromolyn analog” and“cromolyn analogue” (alternative spelling).

Cromolyn derivatives that exhibit improved imaging qualities arepreferred. Cromolyn derivatives of the invention are generallyencompassed by compounds having the formula:

or esters or salts of (I) or (II);

wherein: X is OH, C₁-C₆ alkoxyl, ¹⁸F, or ¹⁹F; Y and Z are independentlyselected from a C₁-C₆ alkyl, C₁-C₆ alkoxyl, halogen, un-substituted orC₁-C₆ substituted amine, ¹⁸F, ¹⁹F, or H; and n is 1, 2, or 3; andwherein for structure (I), if n are both 1 and Y and Z are both H, X isnot OH.

In certain embodiments, X is ¹⁸F or ¹⁹F and, more preferably, Y and Zare hydrogen. A particularly preferred compound has the structure:

or is a salt or ester thereof.

In another embodiment, at least one of Y and Z is ¹⁸F or ¹⁹F and, morepreferably, X is OH. Particularly preferred compounds have thestructures:

or corresponding esters or salts thereof.

In an alternative embodiment, the compound lacks a radiolabel and,preferably, X is OH and Y and Z are hydrogen, except that in such anembodiment comprising structure (I) wherein X is OH and Y and Z arehydrogen, both n cannot be 1.

Preferred compounds of the invention, particularly for imaging purposes,localize to atherosclerotic plaques in the heart, brain and/or carotidartery of a subject.

A preferred dosage range of the present compounds for administration toanimals, including humans, is from about 0.001 mg/kg to about 500 mg/kg.Specifically, for PET and MRI imaging, the preferred dosage range isfrom about 0.1 mg/kg to about 500 mg/kg. Based on these parameters, theartisan may perform no more than routine experimentation to optimize thedosage for a particular application.

Compounds lacking radiolabel are, of course, useful for the treatmentmethods claimed and disclosed herein. Specific methods to synthesizeexemplary compounds according to the invention are set forth below inthe Examples section. In general, the inventors utilize novel syntheticradiofluorination approaches to provide the present compounds. Schemes Iand II shown below illustrate preferred embodiments of the manufacturingprocesses.

In certain embodiments directed to formulations and medicaments fordisease treatment including, e.g., atherosclerosis or Alzheimer's, theinventive compounds may be provided as pharmaceutically acceptablesalts. Other salts may, however, be useful in the preparation of thecompounds according to the invention or of their pharmaceuticallyacceptable salts. Suitable pharmaceutically acceptable salts of thecompounds of this invention include acid addition salts which may, forexample, be formed by mixing a solution of the compound according to theinvention with a solution of a pharmaceutically acceptable acid such ashydrochloric acid, sulphuric acid, methanesulphonic acid, fumaric acid,maleic acid, succinic acid, acetic acid, benzoic acid, oxalic acid,citric acid, tartaric acid, carbonic acid or phosphoric acid.Furthermore, where the compounds of the invention carry an acidicmoiety, suitable pharmaceutically acceptable salts thereof may includealkali metal salts, e.g. sodium or potassium salts, alkaline earth metalsalts, e.g. calcium or magnesium salts; and salts formed with suitableorganic ligands, e.g. quaternary ammonium salts.

The inventive compounds further encompass esters of the describedcompounds, wherein the acidic hydrogen on one or more of the acidicmoieties is substituted by an alkyl group.

Where the compounds according to the invention have at least oneasymmetric center, they may accordingly exist as enantiomers. Where thecompounds according to the invention possess two or more asymmetriccenters, they may additionally exist as diastereoisomers. It is to beunderstood that all such isomers and mixtures thereof in any proportionare encompassed within the scope of the present invention.

The invention also provides pharmaceutical compositions comprising oneor more compounds of this invention in association with apharmaceutically acceptable carrier. Preferably these compositions arein unit dosage forms such as tablets, pills, capsules, powders,granules, sterile parenteral solutions or suspensions, metered aerosolor liquid sprays, drops, ampoules, auto-injector devices orsuppositories; for oral, parenteral, intranasal, sublingual or rectaladministration, or for administration by inhalation or insufflation. Itis also envisioned that the compounds of the present invention may beincorporated into transdermal patches designed to deliver theappropriate amount of the drug in a continuous fashion.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutically acceptable carrier, e.g.conventional tableting ingredients such as corn starch, lactose,sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalciumphosphate or gums, and other pharmaceutical diluents, e.g. water, toform a solid preformulation composition containing a homogeneous mixturefor a compound of the present invention, or a pharmaceuticallyacceptable salt thereof. When referring to these preformulationcompositions as homogeneous, it is meant that the active ingredient isdispersed evenly throughout the composition so that the composition maybe easily subdivided into equally effective unit dosage forms such astablets, pills and capsules. This solid pre-formulation composition isthen subdivided into unit dosage forms of the type described abovecontaining from 0.1 to about 500 mg of the active ingredient of thepresent invention. Typical unit dosage forms contain from 1 to 100 mg,for example, 1, 2, 5, 10, 25, 50 or 100 mg, of the active ingredient.The tablets or pills of the novel composition can be coated or otherwisecompounded to provide a dosage affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich, serves to resist disintegration in the stomach and permits theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol and cellulose acetate.

The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as cottonseedoil, sesame oil, coconut oil or peanut oil, as well as elixirs andsimilar pharmaceutical vehicles. Suitable dispersing or suspendingagents for aqueous suspensions include synthetic and natural gums suchas tragacanth, acacia, alginate, dextran, sodium caboxymethylcellulose,methylcellulose, polyvinylpyrrolidone or gelatin.

The compounds of the present invention are particularly useful whenformulated in the form of a pharmaceutical injectable dosage, includinga compound described and claimed herein in combination with aninjectable carrier system. As used herein, injectable and infusiondosage forms (i.e., parenteral dosage forms) include, but are notlimited to, liposomal injectables or a lipid bilayer vesicle havingphospholipids that encapsulate an active drug substance. Injectionincludes a sterile preparation intended for parenteral use.

Five distinct classes of injections exist as defined by the USP:emulsions, lipids, powders, solutions and suspensions. Emulsioninjection includes an emulsion comprising a sterile, pyrogen-freepreparation intended to be administered parenterally. Lipid complex andpowder for solution injection are sterile preparations intended forreconstitution to form a solution for parenteral use. Powder forsuspension injection is a sterile preparation intended forreconstitution to form a suspension for parenteral use. Powderlyophilized for liposomal suspension injection is a sterile freeze driedpreparation intended for reconstitution for parenteral use that isformulated in a manner allowing incorporation of liposomes, such as alipid bilayer vesicle having phospholipids used to encapsulate an activedrug substance within a lipid bilayer or in an aqueous space, wherebythe formulation may be formed upon reconstitution. Powder lyophilizedfor solution injection is a dosage form intended for the solutionprepared by lyophilization (“freeze drying”), whereby the processinvolves removing water from products in a frozen state at extremely lowpressures, and whereby subsequent addition of liquid creates a solutionthat conforms in all respects to the requirements for injections. Powderlyophilized for suspension injection is a liquid preparation intendedfor parenteral use that contains solids suspended in a suitable fluidmedium, and it conforms in all respects to the requirements for SterileSuspensions, whereby the medicinal agents intended for the suspensionare prepared by lyophilization. Solution injection involves a liquidpreparation containing one or more drug substances dissolved in asuitable solvent or mixture of mutually miscible solvents that issuitable for injection.

Solution concentrate injection involves a sterile preparation forparenteral use that, upon addition of suitable solvents, yields asolution conforming in all respects to the requirements for injections.Suspension injection involves a liquid preparation (suitable forinjection) containing solid particles dispersed throughout a liquidphase, whereby the particles are insoluble, and whereby an oil phase isdispersed throughout an aqueous phase or vice-versa. Suspensionliposomal injection is a liquid preparation (suitable for injection)having an oil phase dispersed throughout an aqueous phase in such amanner that liposomes (a lipid bilayer vesicle usually containingphospholipids used to encapsulate an active drug substance either withina lipid bilayer or in an aqueous space) are formed. Suspension sonicatedinjection is a liquid preparation (suitable for injection) containingsolid particles dispersed throughout a liquid phase, whereby theparticles are insoluble. In addition, the product may be sonicated as agas is bubbled through the suspension resulting in the formation ofmicrospheres by the solid particles.

The parenteral carrier system includes one or more pharmaceuticallysuitable excipients, such as solvents and co-solvents, solubilizingagents, wetting agents, suspending agents, thickening agents,emulsifying agents, chelating agents, buffers, pH adjusters,antioxidants, reducing agents, antimicrobial preservatives, bulkingagents, protectants, tonicity adjusters, and special additives.

The compounds according to the present invention are anticipated to actas treatment agents for inflammation, particularly atheroscleroticplaques, as can be demonstrated by standard protocols commonly known inthe field. Accordingly, another aspect of the invention provides amethod for treating atherosclerotic plaque in a subject, comprisingadministering to a subject an effective dosage of a compound accordingto the present invention, whereby the atherosclerotic plaque is treatedin the subject. In the treatment of atherosclerotic plaque, suitabledosage level (i.e., an effective amount) is from about 0.001 mg/kg toabout 500 mg/kg per day, preferably about 1 mg/kg per day. The compoundsmay be administered on a regimen of 1 to 4 times per day, or on acontinuous basis.

In one embodiment, the present invention is a method for treatingAlzheimer's disease in a subject comprising the step of administering bysystemic delivery an effective amount of a compound having the formula:

or a salt or ester of (I) or (II);wherein: X is OH, C₁-C₆ alkoxyl;Y and Z are independently selected from a C₁-C₆ alkyl, C₁-C₆ alkoxyl,halogen, un-substituted or C₁-C₆ substituted amine, ¹⁸F, ¹⁹F, or H; andn is 1, 2, or 3, wherein Alzheimer's Disease is treated in the subject.

In one specific embodiment, the method of administering by systemicdelivery is selected from the group consisting of oral, parenteral,intranasal, sublingual, rectal, and transdermal administration.

In another embodiment, the present invention is a method for inhibitingthe polymerization of amyloid-beta peptide oligomers in a subjectcomprising the step of administering by systemic delivery an effectiveamount of a compound having the formula:

or a salt or ester of (I) or (II);wherein: X is OH;Y and Z are independently selected from a C₁-C₆ alkyl, C₁-C₆ alkoxyl,halogen, un-substituted or C₁-C₆ substituted amine, ¹⁸F, ¹⁹F, or H; andn is 1, 2, or 3, wherein the polymerization of amyloid-beta peptideoligomers in the subject is inhibited.

In one specific embodiment, the method of administering by systemicdelivery is selected from the group consisting of oral, parenteral,intranasal, sublingual, rectal, and transdermal administration.

In another specific embodiment, the method of inhibiting thepolymerization of amyloid-beta peptide oligomers comprises treatingAlzheimer's disease in the subject.

The Example describes the use of cromolyn and its derivatives forinhibiting polymerization of Alzheimer's Disease oligomers and thustreating Alzheimer's Disease.

The compounds according to the present invention are anticipated to actas treatment agents for Alzheimer's Disease, as can be demonstrated bystandard protocols commonly known in the field. Accordingly, anotheraspect of the invention provides a method for treating Alzheimer'sDisease in a subject, comprising administering to a subject an effectivedosage of a compound of the invention, whereby Alzheimer's Disease istreated in the subject. In the treatment of Alzheimer's Disease,suitable dosage level (i.e., an effective amount) is from about 0.001mg/kg to about 500 mg/kg per day, preferably about 0.1 mg/kg to about 50mg/kg per day, more preferably about 1-10 mg/kg per day. The compoundsmay be administered on a regimen of 1 to 4 times per day, or on acontinuous basis.

In another embodiment, an appropriate dosage level for the presentinvention may generally be about 0.001 to about 500 mg per kg subjectbody weight per day which can be administered in a single or multipledoses. Preferably, the dosage level will be about 0.01 to about 250mg/kg per day; more preferably about 0.05 to about 100 mg/kg per day. Asuitable dosage level may be about 0.01 to about 250 mg/kg per day,about 0.05 to about 100 mg/kg per day, or about 0.1 to about 50 mg/kgper day. Within this range the dosage may be about 0.05 to about 0.5,about 0.5 to about 5 or about 5 to about 50 mg/kg per day. The dosagemay be selected, for example, to include any dose within any of theseranges, for therapeutic efficacy and/or symptomatic adjustment of thedosage to the subject to be treated.

It will be understood that the specific dose level and frequency ofdosage for any particular subject may be varied and will depend upon avariety of factors including the activity of the specific compoundemployed, the metabolic stability and length of action of that compound,the age, body weight, general health, sex, diet, mode and time ofadministration, rate of excretion, drug combination, the severity of theparticular condition, and the subject undergoing therapy.

The following examples are, of course, offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way. Indeed, various modifications of the invention in additionto those shown and described herein will become apparent to thoseskilled in the art from the foregoing description and the followingexamples and fall within the scope of the appended claims.

III. Examples Example 1

Synthesis of F-18 Labeled Cromolyn

Radiofluorination.

5,5′-(2-[18F]fluoropropane-1,3-diyl)bis(oxy)bis(4-oxo-4H-chromene-2-carboxylicAcid)

A Wheaton 5-mL reaction vial containing 50 mCi of fluorine-18 in 1 mL of¹⁸O-enriched water, Kryptofix-2.2.2. (6 mg), and potassium carbonate (2mg) was heated at 120° C. and solvent was evaporated with the aid ofnitrogen gas. The K¹⁸F/Kryptofix complex was dried three times at 120°C. by the addition of 1 mL of acetonitrile followed by evaporation ofthe solvent using a nitrogen flow. A solution of1,3-bis[tolylsulfonyl)oxy]-2-[(trifluoromethyl)sulfonyl]oxy-propane (4mg) in acetonitrile was added to the vial and fluorination was performedat 80° C. for 10 min. The resultant 2-[¹⁸F]fluoropropane 1,3-ditosylatesolution (90% r×n, radioTLC) was passed through a silica gel SepPakusing methylene chloride into a vial containing K₂CO₃ (10 mg) and ethyl5-hydroxy-4-oxo-4H-chromene-2-carboxylate (10 mg). After solventremoval, N-methyl-2-pyrrolidone (NMP) was added and the mixture washeated for 20 min at 140° C. Once cooled, 1 M NaOH (100 uL) was addedand the mixture was heated for 10 min at 80° C. The mixture was dilutedwith 1M HCl (3 mL) and passed through a C-18 SepPak. Polar materialswere eluted with 1M HCl and F-18 cromolyn with 20:80 acetonitrile/PBS (1mL). F-18 cromolyn was purified by HPLC (Phenomenex Luna C18, 250×10 mm,gradient: 0 to 40% acetonitrile in 20 mM phosphate buffer, pH 6.5).Solvent was evaporated the activity (5 mCi, 20% EOB) was dissolved insaline and filtered (0.22μ Millex-GV). Synthesis was complete within 2hr and chemical purity was greater than 95%.

Example 2

Synthesis of Precursors

2-Carbethoxy-5-hydroxy-γ-chromone

A mixture of 2,6-dihydroxyacetophenone (1.0 g, 6.6 mmol) and ethyloxalate (0.15 g, 6.6 mmol) in ether (10 mL) was added to a solution ofsodium ethoxide (0.4 g Na, 20 mmol) in ethanol (15 mL). The mixture wasstirred at 25° C. for 30 min, heated at reflux for 1.5 hr, cooled andfiltered. The precipitated sodium salt was washed with ether and dried.It was then dissolved in water and acidified with 10% HCl to form asticky solid. The solid was refluxed in ethanol (20 mL) with a catalyticamount of 36% HCL for 1 hr. The mixture was poured into 50 mL of waterand extracted twice with methylene chloride (50 mL). The extracts werecombined and dried. After solvent removal, the crude material waschromatographed on silica gel (ethyl acetate/hexane 20:80) to yield 0.57g (40%) of a yellow product; mp 146-148° C. (Lit. 148° C.); ¹H NMR(CDCl₃), d 1.42 (t, 3H, J=7.14 Hz, CH3), 4.47 (q, 2H, J=7.14 Hz), 6.82(d, 2H, J=8.24 Hz, Aro-H), 7.02 (d, 2H, J=4.0 Hz, Aro-H), 7.02 (s, 1H,vinyl-H), 7.58 (t, 1H, J=8.24 Hz, Aro-H), 12.1 (s, 1H, phenol-H).

1,3-Bis(4-methylbenzenesulfonate) propanetriol

A solution of glycerol (11 g, 120 mmol) in methylene chloride (80 mL),DMAP (30 mg) and pyridine (20 mL) was treated at 0-5° C. withp-toluenesulfonyl chloride (54.6 g, 239 mmol) over a period of 30 min.The mixture was stirred at 25° C. for 16 hr, diluted with water (100 mL)and layers separated. The methylene chloride layer was washed with 1NHCl until the wash solution was acidic and then dried (sodium sulfate).After solvent removal, the crude material was chromatographed on silicagel (methanol/methylene chloride 0:100 to 5:95) to yield 14.5 g (30%) ofan oil; ¹H NMR (CDCl₃), δ 2.5 (s, 6H, CH3), 4.25 (m, 4H, CH2), 5.09 (m,1H, CH), 7.4 (d, 4H, J=8.1 Hz, Aro-H), 7.78 (d, 4H, J=8.4 Hz, Aro-H).

1,3-Bis(4-methylbezenesulfonate)-2-trifluoromethylsulfonate propanetriol

A mixture of 1,3-bis(4-methylbezenesulfonate) propanetriol 100 mg (0.25mmol) in methylene chloride (20 mL) and pyridine (1 mL) at 0-5° C. wastreated with trifluoromethanesulfonic anhydride (141 mg, 0.50 mmol). Themixture was stirred at 0-5° C. for 1 hr and then allowed to warm to 25°C. and stirred for 4 hr. The mixture was diluted with water (30 mL) andlayers separated. The methylene chloride layer was washed with 1N HCluntil the wash solution was acidic and then dried (sodium sulfate).After solvent removal, the crude material was chromatographed on silicagel (methylene chloride) to yield 66 mg (50%) of a solid; mp 145-147°C.; ¹HNMR (CDCl₃), δ 2.5 (s, 6H, CH3), 4.25 (d, 4H, J=4.6 Hz, CH2, 5.09(m, 1H, CH), 7.4 (d, 4H, J=8.1 Hz, Aro-H), 7.78 (d, 4H, J=8.4 Hz,Aro-H).

3-Bis(4-methylbezenesulfonate)-2-fluoropropanediol

A solution of 1,3-bis(4-methylbezenesulfonate) propanetriol (2.7 g, 6.78mmol) in methylene chloride (20 mL) at 0-5° C. was treated with DAST(2.18 g, 13.6 mmol). The mixture was stirred at 0-5° C. for 30 thenallowed to warm to 25 and stirred for 16 hr. The mixture was poured intoa sat'd sodium bicarbonate solution (30 mL) and layers separated. Themethylene chloride layer dried (sodium sulfate). After solvent removal,the crude material was chromatographed on silica gel (methylenechloride) to yield 0.82 g (30%) of a solid; mp 99-102° C.; ¹H NMR(CDCl₃), δ 2.5 (s, 6H, CH3), 4.15 (dd, 4H, J=12.3, 4.6 Hz, CH2, 4.8 (dq,1H, J=47, 4.6, CHF), 7.45 (d, 4H, J=8.1 Hz, Aro-H), 7.75 (d, 4H, J=8.4Hz, Aro-H).

Example 3

Synthesis of Standard

5,5′-(2-fluoropropane-1,3-diyl)bis(oxy)bis(4-oxo-4H-chromene-2-carboxylicAcid)

1,3-Bis(2-acetyl-3-hydroxyphenoxy)-2-fluoropropane

A mixture of 3-bis(4-methylbezenesulfonate)-2-fluoropropanediol (1.0,2.5 mmol), 2,6-dihydroxyacetophenone (0.76 g, 5.0 mmol) and potassiumcarbonate (0.69 g) in acetonitrile (40 mL) was heated under reflux for16 hr. The mixture was filtered and the filtrate was evaporated. Thecrude material was chromatographed on silica gel (acetonitrile/methylenechloride 5:95) to yield 0.57 g (40%) of product; mp 162-165° C.; ¹H NMR(d6-DMSO), δ 2.5 (s, 6H, 2CH3), 4.38 (m, 4H, 2CH2), 5.22 (br d 1H, J=49Hz, CHF), 6.45 (m, 4H, 4Aro-H), 7.28 (t, 2H, J=4.55 Hz, 2Aro-H).

1,3-Bis(2-carboxychromon-5-yloxy)-2-fluoropropane diethyl ester

A mixture of 1,3-bis(2-acetyl-3-hydroxyphenoxy)-2-fluoropropane (200 mg,0.52 mmol) and ethyl oxalate (2 mL) was added to a solution of sodiumethoxide (87 mg Na) in ethanol (10 mL) and benzene (10 mL). The mixturewas heated at reflux for 16 hr, cooled and diluted with ether (50 mL).The precipitated sodium salt was filtered, washed with ether and dried.It was then dissolved in water and acidified with 10% HCl to obtain asticky solid. The solid was refluxed in ethanol (20 mL) with a catalyticamount of 36% HCL for 1 hr. The mixture was poured into 50 mL of waterand extracted twice with methylene chloride (50 mL). The extracts werecombined and dried. After solvent removal, the crude material waschromatographed on silica gel (acetonitrile/methylene chloride 10:90) toyield 0.12 g (45%) of a white product; mp 166-170° C.; ¹H NMR (CDCl₃), d1.42 (t, 6H, J=7.14 Hz, 2CH3), 4.58 (q, 4H, J=7.14 Hz 2CH2), 4.65 (m,4H, 2CH2), 5.35 (dq, 1H, J=46 Hz, J=4.4 HZ, CHF), 6.90 (s, 2H, vinyl-H),6.95 (d, 2H, J=8.24 Hz, 2Aro-H), 7.13 (d, 2H, J=8.24 Hz, 2Aro-H) 7.6 (t,2H, J=8.24 2Aro-H).

5,5′-(2-fluoropropane-1,3-diyl)bis(oxy)bis(4-oxo-4H-chromene-2-carboxylicAcid)

A suspension of 1,3-bis(2-carboxychromon-5-yloxy)-2-fluoropropanediethyl ester (100 mg, 0.19 mmol) in methanol (20 mL) and 1 M sodiumhydroxide (2 mL) was heated at 80 C for 1 hr. The solution was acidifiedwith 10% HCl and volatiles were removed. A solution ofmethanol/methylene chloride (50:50) was added to the solid and themixture was filtered. Evaporation afforded 76 mg (85%) of product; ¹HNMR (d6-DMSO), δ 4.65 (m, 4H, 2CH2), 5.32 (br d, 1H, J=46 Hz, CHF), 6.80(s, 2H, 2vinyl-H), 7.2 (d, 2H, J=8.24 Hz, 2Aro-H), 7.71 (t, 2H, J=8.242Aro-H).

Example 4

Biodistribution

Biodistribution of F-18 cromolyn (Compound A) was performed in normalmice at 5, 30 and 60 min after intravenous injection into the tail vein(50 uCi per animal). At 5 min, organ activity (DPG) was: heart: 1.09%,blood: 3.3%, lung: 1.90%, liver: 7.69%, and brain: 0.15%. At 30 and 60min, washout of activity was seen in all organs. Heart uptake wasdecreased from 1.09% to 0.18%. The data is provided in Table 1 below andin bar graph format in FIG. 4.

TABLE 1 F-18 Cromolyn Tissue Distribution (% Dose/gram) in Normal MiceOrgan 5 min 30 min 60 min blood 3.31 ± 1.37 0.92 ± 0.20  0.44 ± 0.15 heart 1.09 ± 0.13 0.32 ± 0.03  0.18 ± 0.07  lung 1.90 ± 1.04 0.62 ±0.19  0.29 ± 0.21  liver 7.69 ± 0.41 3.11 ± 0.36  1.76 ± 0.74  brain0.15 ± 0.37 0.04 ± 0.007 0.03 ± 0.004 Standard deviation (±)

Example 5

Aromatic Radiofluorination

Radiofluorination of cromolyn N,N,N-trimethylbenzenaminium triflate isdone in a sealed vial containing dry K¹⁸F/Kryptofix in dimethylacetamide(DMAc) for 5 min at 140° C. The resultant F-18 cromolyn solution isdiluted with water and passed through a C-18 SepPak. Polar materials areeluted with water and product eluted with methanol into a vial. Asolution of 1N sodium hydroxide is added and the vial is heated for 10min at 80° C. The mixture is acidified and F-18 cromolyn is purified byHPLC.

Alternative Route:

Example 6

Synthesis of Precursors

1-(4-Amino-2,6-dimethoxyacetophenone) [Dillon, Michael Patrick;Jahangir, Alam; Moore, Amy Geraldine; Wagner, Paul J. U.S. Pat. Appl.Publ. (2007), 49 pp] Step 1.N-(3,5-Dimethoxyphenyl)-2,2,2-trifluoroacetamide

To 3,5-dimethoxyaniline (20 g, 131 mmol) dissolved in anhydroustetrahydrofuran (90 mL) was added 4-(dimethylamin) pyridine (1.6 g, 13.1mmol) and ethyl trifluoroacetate (47 mL, 392 mmol). After refluxing 48hours, the cooled reaction mixture was concentrated and partitionedbetween ethyl acetate (300 mL) and 2N hydrochloric acid (100 mL). Theethyl acetate layer is washed with water (100 mL), dried using anhydroussodium sulfate, and concentrated to yieldN-(3,5-dimethoxyphenyl)-2,2,2-trifluoroacetamide (31.8 g, 98%) as a paleyellow solid.

Step 2. N-(4-Acetyl-3,5-dimethoxyphenyl)-2,2,2-trifluoroacetamide

To a solution of N-(3,5-dimethoxyphenyl)-2,2,2-trifluoroacetamide (31.8g, 130 mmol) in anhydrous methylene chloride (450 mL), cooled in an icebath, is added a solution of tin (IV) chloride (29.9 mL, 260 mmoldissolved in 30 mL anhydrous methylene chloride) dropwise over 10minutes. Acetyl chloride (9.1 mL, 130 mmol) is added slowly, maintainingthe temperature of the reaction below 5° C. After stirring 3 hours atroom temperature, the reaction is cooled in an ice bath. Water (300 mL)is added, maintaining the temperature of the reaction below 25° C., andthe reaction is stirred at room temperature for 18 hours. The reactionmixture is extracted with methylene chloride, and the organic layer isseparated, washed with water, dried, filtered and evaporated underreduced pressure. The residue is purified by silica gel columnchromatography eluting with 20% to 30% hexanes/ethyl acetate to yieldN-(4-acetyl-3,5-dimethoxyphenyl)-2,2,2-trifluoroacetamide (4.8 g, 13%)as a white solid.

Step 3. 1-(4-Amino-2,6-dimethoxyacetophenone)

To N-(4-Acetyl-3,5-dimethoxyphenyl)-2,2,2-trifluoroacetamide (4.3 g,14.8 mmol) dissolved in methanol (90 mL) is added anhydrous potassiumcarbonate (4.67 g, 33.8 mmol). After refluxing for 18 hours, thereaction mixture is cooled and concentrated under reduced pressure. Theconcentrate is extracted with ethyl acetate and the organic layer waswashed with brine, dried, filtered and concentrated under reducedpressure to yield 4-amino-2,6-dimethoxyacetophenone (2.5 g, 87%) as apale yellow solid.

4-Dimethylamino-2,6-hydroxyacetophenone [Brooks P R, Wirtz C, et al. J.Org. Chem. 1999, 64:9719-21]

4-Amino-2,6-dimethoxyacetophenone and n-tetrabutylammonium iodide isstirred in methylene chloride at −78 C. A solution of boron trichloridein methylene chloride is added and the solution is then stirred at 0° C.for 1 hr. The reaction is quenched with ice-water and stirred for 30min, diluted with saturated sodium bicarbonate, and extracted withmethylene chloride. The combined extracts are dried and purified bychromatography on silica gel to provide4-dimethylamino-2,6-hydroxyacetophenone.

2-Carbethoxy-3-dimethylamino-5-hydroxy-γ-chromone ethyl ester or2-Carbethoxy-4-hydroxy-γ-chromone ethyl ester

A mixture of 4-dimethylamino-2,6-hydroxyacetophenone (or2,5-dihydroxyacetophenone) (Sigma-Aldrich) and ethyl oxalate in ether isadded to a solution of sodium ethoxide in ethanol. The mixture isstirred at 25° C. for 30 min, heated at reflux for 1.5 hr, cooled andfiltered. The precipitated sodium salt is washed with ether and dried.It is then dissolved in water and acidified with 10% HCl to form asticky solid. The solid is refluxed in ethanol (20 mL) with a catalyticamount of 36% HCL for 1 hr. The mixture is poured into 50 mL of waterand extracted twice with methylene chloride (50 mL). The extracts arecombined and dried. After solvent removal, the crude material ischromatographed on silica gel (ethyl acetate/hexane 20:80) to yield2-carbethoxy-3-dimethylamino-5-hydroxy-γ-chromone ethyl ester (or2-carbethoxy-4-hydroxy-γ-chromone ethyl ester).

A mixture of 1,3-bis[(tolylsulfonyl)oxy]-2-propanol (1 equivalent),2-carbethoxy-5-hydroxy-γ-chromone ethyl ester (or2-carbethoxy-5-hydroxy-γ-chromone ethyl ester) (1 equivalent) andpotassium carbonate in N-methyl-2-pyrrolidone (NMP) is heated for 6 hrat 140° C. Once cooled, the mixture is diluted with water and extractedwith methylene chloride. Extracts are combined and dried. After solventremoval, the crude material is chromatographed on silica gel to yieldproduct.

A mixture of 2-carbethoxy-3-dimethylamino-5-hydroxy-γ-chromone ethylester (1 equivalent), the appropriate 2-propanol tosylate above andpotassium carbonate in N-methyl-2-pyrrolidone is heated for 6 hr at 140°C. Once cooled the mixture is diluted with water and extracted withmethylene chloride. Extracts are combined and dried. After solventremoval, the crude material is chromatographed on silica gel to yieldproduct.

Example 7

Acetylation of Cromolyn Derivatives

A solution of the cromolyn derivative in methylene chloride and pyridineis treated with acetic anhydride at 0° C. and then allowed to warm to25° C. where it is stirred for 4 hr. The mixture is washed with 10%sodium bicarbonate and dried. The solvents are removed by vacuum and thecrude solid is purified by chromatography.

Example 8

Preparation of Trimethyl Ammonium Triflate Salts

A solution of the acetylated cromolyn derivative in methylene chlorideis treated with trifluoromethylsulfonic anhydride at 25° C. for 4 hr.The resultant salt is filtered and washed with methylene chloride.

Example 9

Cromolyn derivative (Compound A) inhibits polymerization of Alzheimer'sDisease oligomers.

One of the common goals in treating Alzheimer's Disease is to eliminateor reduce the AB oligomers that are the neuron toxins. Achieving thisgoal slows down Alzheimer's manifestation. One way to demonstrate theefficacy of a drug to treat Alzheimer's is to test for the inhibition ofthe polymerization of the AB oligomers.

The study described in this example was based on the assay described byFindeis, et al. “Modified-peptide inhibitors of amyloid beta-peptidepolymerization.” Biochemistry 1999, 38 (21), 6791-800.

AB Peptide: 50 μM of HCl salt or Test compound (Compound A): 50 μM

Buffer: 10 mM sodium phosphate, 100 mM NaCl, pH 7.4

Readout polymerization at: OD 405 nm

The results showed that amyloid beta-peptide polymerization in thepresence of cromolyn derivative (Compound A) is 2.5 times slower thanamyloid beta-peptide polymerization in the presence of a controlvehicle.

TABLE 2 Experimental results Compound Time Relative increase Vehicle (noAdd) 7.97 (1.0) TS734 (Compound A) 19.9 2.5 *Time is elapsed time to 50%of maximum signal *Relative increase is time(sample)/time(vehicle)

The data above indicate that exemplary Compound A exhibits appreciablebrain uptake and clearance and, in terms of efficacy, inhibits ABpeptide polymerization. Compound A and related chemical entities aretherefore useful for treatment of Alzheimer's Disease in human subjects.

Example 10 Synthesis of5,5′-(2-[18F]fluorotrimethylenedioxy)bis(4-oxochromene-2-carboxylicacid) sodium salt

5,5′-(2-Hydroxytrimethylenedioxy)bis(4-oxochromene-2-carboxylic acid)diethyl ester

A suspension of cromolyn sodium salt 1 (161 mg, 0.31 mmol) in ethanol(25 mL) and cone. HCl (1 mL) was heated in a sealed flask for 28 h at95-100° C. The suspension dissolved to give a clear colorless solution.Solvent was evaporated and the crude oil was chromatographed on silicagel using 100% ethyl acetate to yield the diethyl ester 2 (132 mg, 80%):TLC Rf=0.44 (100% ethyl acetate); 1HNMR (CDCb, 300 MHz) 8 1.42 (t, 3H,J=7.1 Hz, CH3), 2.73 (br s, 1H, OH), 4.44 (q, 4H, J=7.1 Hz, 20CH2CH3),4.324.59 (m, 5H, CHOH, 20CH2), 6.93-6.99 (rn, 4H, 2 vinyl H, 2 aromaticH), 7.16 (d, 2H, J=8.4 Hz, aromatic H), 7.59 (t, 2H, J=8.2 Hz, aromaticH)

1,3-bis(2-(ethoxycarbonyl)-4-oxo-4H-chromen-5-yloxy)propan-2-ylmethanesulfonate

A solution of the alcohol 2 (107 mg, 0.20 mmol) and triethylamine (41mg, 0.41 mmol) in dichlormethane (25 mL) cooled to 0° C. was treatedwith methanesulfonyl chloride (34 mg, 0.30 mmol). After stirring for 2 hat 0° C., methylene chloride (100 mL) was added and the mixture waswashed with satd. NaHC03 (2×30 mL) and brine (50 mL), dried over MgS04,and concentrated. The residue was purified by flash columnchromatography (80% ethyl acetate in hexane) to give the product 3 (102mg, 84%): TLC R.=0.54 (ethyl acetate); 1H NMR (CDCI3, 300 MHz 8 1.39 (t,3H, J=7.1 Hz, CH3), 3.35 (s, 3H, CH3S02), 4.41 (q, 4H, J=7.2 Hz,20CH2CH3), 4.55-4.66 (m, 4H, 20CH2, 5.40 (quintet, 1H, J=5.0, CHOMs),6.89 (s, 2H, vinyl H), 6.98 (d, 2H, J=8.4 Hz, aromatic H), 7.16 (d, 2H,J=8.8 Hz, aromatic H), 7.61 (t, 2H, J=8.2 Hz, aromatic H); 13C NMR(CDCb, 75 MHz, Ib=1.0 Hz) 8: 14.3, 38.6, 63.1, 68.7, 77.9, 108.7, 111.8,115.6, 116.4, 135.2, 150.7, 158.0, 160.7, 177.7.

5,5′-(2-[18F]Fluorotrimethylenedioxy)bis(4-oxochromene-2-carboxylicacid) sodium salt

A Wheaton 5-mL reaction vial containing fluorine-18 (100 mCi) in 1 mL180-enriched water, and ammonium hydroxide (100 ul) was heated at 120°C. and water was evaporated with the aid of a nitrogen gas flow. Thecontents were dried by the addition of 1 mL of acetonitrile followed byevaporation of solvent using a nitrogen flow. This process is repeatedthree times. A solution of 3 mg of mesylate 3 in 0.1 mL of acetonitrilewas added to the sealed vial and fluorination was performed at 170° C.for 10 min. Once cooled to room temperature, the reaction mixture waspassed through a silica gel Sep-Pak using methylene chloride (3 mL) andthe solvent was removed using a nitrogen flow. A mixture of 0.5 mL 1 Mlithium hydroxide and 1 mL methanol was added to the reaction vial andthe vial heated at 80° C. for 20 min. Solvent was removed and15,5′-(2-[18F]fluorotrimethylenedioxy)bis(4-oxochromene-2-carboxylicacid) sodium salt was purified on a C18 Sep-Pak using PBS, ph 7, and thesolution was filtered (MillexGV 0.22 urn). Radiochemical yield rangedfor 5 to 20% EOB.

Example 11

Synthetic Route for Asymmetric Cromolyn

This example illustrates the inventors' general route for synthesis ofasymmetric cromolyn derivatives.

Example 12

In Vivo Experiments of Cromolyn and Ibuprofen Combination Treatment.

Three mice groups (five animals in each) were tested in a Morris waternavigation test. Two groups were four months young APP/PS1 including amutant Aβ mouse and a model indicative of Alzheimer's Diseaseprogression. One APP/PS1 group was treated with Cromolyn and ibuprofencombination for six months, and the second was untreated as an controlgroup and a third untreated wild type was used as a normal control. FIG.1 is a graph showing the in-vivo study summary. WT (wild type, rightpanel) shows normal untreated mice. The control group (left panel) showstransgenic mice that did not received drug treatment. The treated group(Mid panel) shows transgenic mice that receivedAZLT-OP1(cromolyn+ibuprofen) for six month by Intraperitoneal (IP)injection twice weekly. Mice were trained for 7 days to remember thelocation of the platform. At day 8, the platform was removed, and thetimes of crossing the platform area was recorded.

In another study, 7.5 month old APP/PS1 mice completed treated for aweek as an acute treatment using three different doses of CromolynSodium (1.05 mg/kg, 2.1 mg/kg and 3.15 mg/kg). The treatment was givenby IP injection everyday for 7 days before sacrificing the mice andharvesting the brain. Brain extracts were quantified for the totalamount of Aβ40, Aβ42 and Aβ oligomers.

Here are the main conclusions of this acute study:

1. A dose-dependent decrease in the amount of Aβ40 and Aβ42 associatedwith the two higher doses (2.1 mg/kg and 3.15 mg/kg), up to 50% wasobserved.

2 This effect was sustained after treatment of the samples withguanidine-HCl to dissolve any amyloid aggregates.

3 The quantification of oligomeric species using the 82E1/82E1 ELISA kitfailed to show any significant difference among the experimental groups.

One explanation to the insignificant change is that acute exposure toCromolyn Sodium treatment primarily affects monomeric species, impactingoligomers or higher-order aggregates chronic longer treatment term.Acute treatment would not cause a substantial change in the oligomericquantities.

Example 13

In another experiment, cromolyn derivatives were tested as inhibitors ofAβ polymerization. Inhibiting Aβ oligomer production will provide ofAlzheimer's Disease and treating Alzheimer's Disease.

The investigational product ALZT-OP1a (cromolyn sodium) is a syntheticchromone derivative that has been approved for use by the FDA since the1970s for the treatment of asthma. For asthma treatment, cromolyn sodiumpowder was micronized for inhalation to the lungs via dry powderinhaler, i.e. the Spinhaler device. Liquid intranasal and ophthalmicformulations have also been developed for the treatment of rhinitis andconjunctivitis.

The mechanism of action for cromolyn sodium (ALZT-OP1a) is characterizedas a mast cell stabilizer, namely to suppress cytokine release fromactivated lymphocytes together with preventing the release of histaminefrom mast cells (Netzer, 2012; Keller, 2011). It was administered fourtimes daily as prophylaxis for allergic and exercise-induced asthma, notas a treatment for acute attacks.

Applicants have discovered a new mechanism of action for cromolyn,which, along with its role for suppressing immune responses, enables there-purposing of this approved drug for use to halt AD progression. TheApplicants' studies have shown that cromolyn sodium binds tobeta-amyloid peptides and inhibits its polymerization into oligomers andhigher order aggregates. The inhibition of beta-amyloid polymerizationwill arrest amyloid-mediated intoxication of neurons and restore thepassage of these aberrant beta-amyloid oligomers out of the brain ratherthan their accumulation.

Applicants' studies showed that cromolyn or its derivatives penetratesthe blood-brain barrier in animal models, so that plasma bioavailabilityfollowing cromolyn inhalation will translate to concentrations in thebrain sufficient to interfere with beta-amyloid oligomerization andaccumulation. Inhalation of cromolyn sodium was shown to be the mosteffective non-injected administration route for systemic bioavailabilityof cromolyn sodium in animals and humans (Moss, 1970; Neale, 1986;Richards, 1987; Aswania, 1999; Tronde, 2003). An FDA-approved route ofadministration for cromolyn sodium is oral inhalation using acapsule-based dry powder inhaler, with 20 mg cromolyn sodium loaded percapsule. Studies have shown that with high inspiratory rates, theinhaled cromolyn sodium is delivered efficiently to the human lung, with10-15% of the inhaled drug-delivered-dose absorbed into the bloodstream(Richards, 1987; Keller, 2011). For these reasons, cromolyn sodiuminhalation with a dry powder inhaler device was selected as the route ofadministration in the present invention. However, plasma levels ofcromolyn following inhalation are reported to show high intra- andinter-subject variability, and that cromolyn uptake by asthmatics waslower than in healthy volunteers (Richards, 1987; Keller, 2011).

For planned human studies, each blister will contain the active productingredient (cromolyn sodium) and inhalation grade lactose monohydrate asan excipient. The once-daily cromolyn dose to be tested in this study isless than 20% the dose from the four-times daily approved dose level (80mg cromolyn sodium total per day) for the treatment of asthma.

Taken together, the once daily ALZT-OP1a dose in this study shouldpreserve the drug's excellent safety and tolerability profile, yet ispredicted to achieve the nanomolar drug concentrations needed to blockbeta-amyloid oligomerization in the brain to prevent Alzheimer's diseaseprogression.

Example 14

Cromolyn Derivatives for Inhibiting Polymerization of Alzheimer'sDisease Oligomers.

FIG. 2A and FIG. 2B illustrate the measurement of TBS soluble Aβ levelby WAKO ELISA. The experiments show that TBS Aβ level decreasesfollowing by treatment of cromolyn sodium with dose-dependency. FIG. 2Ashows that Aβ40 level decreases following by treatment of cromolynsodium with dose-dependency. FIG. 2B shows that Aβ42 level decreasesfollowing by treatment of cromolyn sodium with dose-dependency. Asindicated, the number of animals per group is N=3 or 5, average±SE. Thep value is significant using one-way ANOVA test (Bonferroni's test).Both of total soluble Aβ [as shown as Gdn+(Guanidine-HCl)] and monomericAβ [as shown as Gdn−(no guanidine)] decease after the addition ofcromolyn sodium. The dose of 2.1 mg/kg of cromolyn sodium was enough todecrease TBS soluble Aβ.

FIG. 3A, FIG. 3B, and FIG. 3C illustrate the measurement of TBS solubleAβ oligomer level IBL oligomer ELISA (82E1-82E1). The experiments showthat Aβ oligomer level was not changed following the treatment ofcromolyn sodium. FIG. 3A shows the experiments of IBL Aβ oligomer ELISA(82E1-82E1). FIGS. 3B and 3C show the difference the experiments withGdn and those without Gdn using Aβ WAKO ELISA. N=3 or 5 animals pergroup, average±SE. The p value is not significant using one-way ANOVAtest (Bonferroni's test). Both ELISA (IBL oligomer ELISA and thedifferences between with and without Gdn using WAKO ELISA) showed thatoligomer level was not changed following the treatment of cromolynsodium.

Example 15

Discussion

Applicants summarize the rationale behind the treatment utility ofcromolyn as follows:

1. Molecular structure is similar to some that had affinity to plaque(Formula III and table 3). The significant difference is that the drugin the present invention works in nanomolar concentrations as comparedto micromolar concentrations of other previous drugs.

TABLE 3 The structural similarity of fisetin analogues and their effectson Aβ fibril formation. Effects on Substituents Aβ fibril Compound 3 5 73′ 4′ 5′ formation Fisetin OH H OH OH OH H Inhibitory 3′,4′,7- H H OH OHOH H Inhibitory Trihydroxy- flavone 3,3′,4′- OH H H OH OH H InhibitoryTrihydroxy- flavone 3,3′,7- OH H OH OH H H Enhancing Trihydroxy- flavone5-Deoxykaemp- OH H OH H OH H Enhancing ferol Luteolin H OH OH OH OH HInhibitory Quercetin OH OH OH OH OH H Inhibitory Chrysin H OH OH H H HEnhancing Kaempferol OH OH OH H OH H Enhancing Myricetin OH OH OH OH OHOH Inhibitory

2. The suitable molecular weight of the molecules in the presentinvention allows the molecules to penetrate brain.

Chemical Structure:

Molecular Formula: C₂₃H₁₄Na₂O₁₁Molecular Weight: 512.34 [g/mol]

3. The molecules in the present invention have desirable lipophilicity(Log P) and pressure surface area (PSA) in the brain penetration range(Table 4). The PIB analog TS3124 has a 4% brain concentration, and ahigher Log P value in a range that there is no usal uptake. This isbalanced by the much lower PSA. Log P was determined by Chemdraw pro,Version 10. PSA was determined by the previous methods(http://www.daylight.com/meetings/emug00/Ertl/tpsa.html).

TABLE 4 The moelcular structures, molecular weight, lipophilicity (LogP)and pressure surface area (PSA). compound Structure Mw logP PSA PKaTS734

466.41 2.1 127.20 C0399

508.38 1.39 for diacid 125.43 TS3124

302.37 3.92 45.15 OH: 9.2 NH: 19.2

4. Mice biodistribution of radiolabeled cromolyn biodistribution shows1% dose per gram brain accumulate. FIG. 4 illustrates thebiodistribution of radiolabeled cromolyn Compound A followingintravenous injection in mice. In FIG. 4, a 5, 30 or 60 minute,corresponding to Series 1, 2 or 3, respectively in the graph, brainuptake shows 1% accumulation with little or no washout for the periodmeasured.

5. The binding of cromolyn to Aβ and its polymerization inhibition wasconfirmed by four independent methods.

UV Aggregation Assay.

Aβ peptide aggregation and the impact of drugs to slow or prevent Aβaggregation was measured by a UV absorbance assay (Findeis, 1999). Aβ(1-40) peptides, at 50 μM, were mixed with 50 μM drug in assay bufferand the plate was incubated at ambient temperature on a plate reader.The UV absorbance was monitored at 540 nm over a 2-3 h period.

Polymerization of Aβ-monomer peptides into clusters of trimers andtetramers initiates the Aβ aggregation process into protofibrils andthen into fibrils that form amyloid plaques. The polymerizationexperiments revealed that Aβ monomer reached 50% polymerization in 14minutes. At equimolar concentrations with Aβ, the addition of cromolyninhibited the rate of Aβ polymerization 7-fold, namely 50%polymerization required 75 minutes incubation, compared to 14 minutes inthe absence of drug.

TABLE 5 Cromolyn inhibits Aβ polymerization. Relative Increase in %Thioflavin T Polymerization Test Compound Bound Relative Binding Time(fold) Vehicle 37% 1 1 TS734 (cromolyn) 30% 0.82 7.8

LC/MS/MS Binding Assay.

Binding was measured by equilibrium dialysis. Amyloid fibrils werepreformed by incubating the peptide in buffer with shaking for 120 hoursat 27° C. The drugs were incubated with fibrils (50 μM peptide) in a REDequilibrium dialysis device (Pierce), and the amount of test agent oneach side is determined by LC/MS/MS. Percent bound was calculated as1−(free conc/total conc) after correcting for background signal.Thioflavin-T was used as a positive control. Binding is displacement ofThioflavin T. Polymerization is ranked for relative Aβ. In general,compounds that rank highly in inhibiting polymerization rank low inbinding to aggregates, and vice versa.

Competition Binding Assay.

The competition assay was performed as described previously (Ono andHayashi, 2009). Amyloid peptide aggregates were preformed by incubatingAβ (1-40) peptide with buffer for 3 days at 37° C. Drugs at 20 μM weremixed with assay solution containing 10 μg/mL amyloid peptideaggregates+3 μM Thioflavin-T on one side of a RED dialysis device withassay buffer added to the other side. After 4 h dialysis, the amount ofThioflavin-T was determined by LC/MS/MS. The relative binding wasdetermined by normalizing the percent binding by the percent binding ofthe vehicle control.

Aβ Aggregation by Thioflavin T Assay.

One of the most routinely used approaches to monitor Aβ polymerizationis the thioflavin T binding assay. When thioflavin T binds to beta-sheetrich structures, such as amyloid aggregates, the dye displays enhancedfluorescence and a characteristic red shift in its emission spectrum. Aβpeptide at 5 μM was mixed with 10 μM thioflavin T with drug at differentconcentrations. In the absence of drug, Aβ polymerization showsincreasing thioflavin T fluorescence over 60-180 min, as shown in FIG.4.

The addition of cromolyn (CO399) and its ¹⁸F derivative (TS734) atnanomolar concentration shows inhibition of Aβ aggregation, as shown inFIG. 5.

By four separate in vitro assays, cromolyn sodium, at nanomolarconcentrations, effectively inhibits Aβ amyloid peptide polymerizationinto oligomers and higher order aggregates.

6. Preliminary analysis of the binding model indicates that cromolynbinding to the surface of beta sheet across the beta strand in a mannersimilar to Thioflavin-T. FIGS. 6 and 7 illustrate the side and top viewof the relative structures and locations of cromolyn and Aβ aftercromolyn binds Aβ through a binding model simulation.

7. Applicants tested several other structures for treating AD inaddition to cromolyn. Several types of compounds for both imaging andtherapeutic agents have been evaluated for Aβ peptide polymerizationinhibition.

In an effort to combine bioavailability and dual function, Applicantshave tethered scyllo-inositol, which is transported across theblood-brain barrier and known to bind and neutralize oligomers intosoluble complexes (McLaurin, Kierstead, et al., 2006; Sun, Zhang, etal., 2008), to 2-ethyl-8-methyl-2,8-diazospiro-4,5-decan-1,3-dione, amuscarinic M1 receptor agonist (Palacios, Bolliger, et al., 1986). RS-86was chosen because evidence has shown that it improves cognitivefunction, mood and social behavior in some AD patients (Wettstein andSpiegel, 1985). M2 receptors function in cholinergic nerve terminals toregulate the release of acetylcholine, whereas M1 receptors are locatedon postsynaptic cells and facilitate cellular excitation (Mash, Flynn,1985). Since presynaptic cholinergic neurons degenerate in AD whilepostsynaptic M1 muscarinic receptors remain in tact, the use oflong-acting muscarinic agonists like RS-86 has been proposed as atreatment strategy for memory loss. However, RS86 has low brainpenetration; combining it with inositol using a linkage which can bemetabolized once in the brain may increase bioavailability of theagonist as well as maintaining the beneficial effect of inositol. In thepast, both inositol, in the form of 1-fluoro-scyllo-inositol, and RS-86derivatives have been radiolabeled with F-18 or C-11 as potential PETprobes for AD.

8. It is believed that these suitable compounds target mast cells byinhibiting cytokine production therefore an additional treatment theinflammatory response associated with the AD trigger and process. Intheir previous publication (Jin, Silverman, et al. 2009), Jin andco-workers indicate that the potential cromolyn compounds can be used asa Mast cell inhibitors.

Example 16

Non-Steroidal Anti-Inflammatory Drugs (NSAIDs).

Compelling evidence from multiple epidemiology studies revealed thatlong-term dosing with non-steroidal anti-inflammatory drugs (NSAIDs)dramatically reduced AD risk in the elderly, including delayed diseaseonset, reduced symptomatic severity and slowed cognitive decline (Veld,2001; Etminan, 2003; Imbimbo, 2010). Three mechanisms have been proposedhow NSAIDs inhibit the processes that contribute to AD progression: i)by inhibiting COX activity to reduce or prevent microglial activationand cytokine production in the brain (Mackenzie, 1998; Alafuzoff, 2000;Yan, 2003; Gasparini, 2004; Imbimbo, 2010); ii) by reducing amyloiddeposition (Weggen, 2001; Yan, 2003; Imbimbo, 2010); or iii) by blockingCOX-mediated prostaglandin E2 responses in synapses (Kotilinek, 2008).

Therefore, NSAIDs are predicted to dampen the neuro-inflammatoryresponse and impact AD progression via several mechanisms. Whenadministered together with drugs that inhibitbeta-amyloidoligomerization, the combination treatment paradigm isproposed to attenuate the multiple triggers leading to neurodegenerationand neuronal death. The decline in cognitive performance may bereversed, due to neuronal plasticity and neurogenesis in the hippocampus(Kohman, 2013), if AD progression is arrested at a very early stage.

Ibuprofen.

Ibuprofen is a non-selective COX inhibitor for treating inflammation asa non-steroidal anti-inflammatory drug (NSAID). The COX enzymes convertcertain fatty acids to prostaglandins. The prostaglandins at the end ofthe “chain” of reactions that starts with the COX enzyme cause anincreased sensitivity to pain, fever, and vasodilation (increased bloodflow or inflammation). By inhibiting the start of this chain ofreactions, ibuprofen therefore reduces pain, fever, and inflammation.Because ibuprofen blocks the activity of both COX enzymes, it isconsidered a non-selective COX inhibitor NSAID.

ALZT-OP1 therapy for the treatment of individuals with amnestic mildcognitive impairment. ALZT-OP1 is a multi-functional drug therapyconsisting of cromolyn sodium (ALZT-OP1a) administered by inhalation toinhibit beta-amyloid peptide polymerization and to dampen immuneresponses, plus a concomitant but separately administered low dose oralibuprofen tablet (ALZT-OP1b) to inhibit the neuro-inflammatory responsein persons with confirmed amnestic mild cognitive impairment (aMCI) dueto Alzheimer's disease. Both active pharmaceutical ingredient (API)drugs in this ALZT-OP1 formulation are approved, marketed drugs thathave been re-purposed for use to prevent the onset of dementia andAlzheimer's disease progression.

ALZT-OP1a

The investigational product ALZT-OP1a (cromolyn sodium) is a syntheticchromone derivative that has been approved for use by the FDA since the1970s for the treatment of asthma. For asthma treatment, cromolyn sodiumpowder was micronized for inhalation to the lungs via dry powderinhaler, i.e., the Spinhaler device. Liquid intranasal and ophthalmicformulations have also been developed for the treatment of rhinitis andconjunctivitis.

The mechanism of action for cromolyn sodium (ALZT-OP1a) is characterizedas a mast cell stabilizer, namely to suppress cytokine release fromactivated lymphocytes together with preventing the release of histaminefrom mast cells (Netzer, 2012; Keller, 2011). It was administered fourtimes daily as prophylaxis for allergic and exercise-induced asthma, notas a treatment for acute attacks.

We have discovered a new mechanism of action for cromolyn, which, alongwith its role for suppressing immune responses, enables the re-purposingof this approved drug for use to halt AD progression. Our studies haveshown that cromolyn sodium binds to beta-amyloid peptides and inhibitsits polymerization into oligomers and higher order aggregates. Theinhibition of beta-amyloid polymerization will arrest amyloid-mediatedintoxication of neurons and restore the passage of these aberrantbeta-amyloid oligomers out of the brain rather than their accumulation.

Our studies showed that cromolyn penetrates the blood-brain barrier inanimal models, so that plasma bioavailability following cromolyninhalation will translate to concentrations in the brain sufficient tointerfere with beta-amyloid oligomerization and accumulation. Inhalationof cromolyn sodium was shown to be the most effective non-injectedadministration route for systemic bioavailability of cromolyn sodium inanimals and humans (Moss, 1970; Neale, 1986; Richards, 1987; Aswania,1999; Tronde, 2003). An FDA-approved route of administration forcromolyn sodium is oral inhalation using a capsule-based dry powderinhaler, with 20 mg cromolyn sodium loaded per capsule. Studies haveshown that with high inspiratory rates, the inhaled cromolyn sodium isdelivered efficiently to the human lung, with 10-15% of the inhaleddrug-delivered-dose absorbed into the bloodstream (Richards, 1987;Keller, 2011). For these reasons, cromolyn sodium inhalation with a drypowder inhaler device was selected as the route of administration inthis study. However, plasma levels of cromolyn following inhalation arereported to show high intra- and inter-subject variability, and thatcromolyn uptake by asthmatics was lower than in healthy volunteers(Richards, 1987; Keller, 2011).

Cromolyn sodium powder blend (ALZT-OP1a) will be loaded into blistersfor use with a dry powder inhaler with reproducible aerosol performanceat a range of inspiratory rates. Each blister will contain the activeproduct ingredient (cromolyn sodium) and inhalation grade lactosemonohydrate as an excipient. The once-daily cromolyn dose to be testedin this study is less than 20% the dose from the four-times dailyapproved dose level (80 mg cromolyn sodium total per day) for thetreatment of asthma. The dose is calculated to titrate the estimateddaily 22-27 nanogram of Aβ amyloid plaque produced in the brain.

Taken together, the once daily ALZT-OP1a dose in this study shouldpreserve the drug's excellent safety and tolerability profile, yet ispredicted to achieve the nanomolar drug concentrations needed to blockbeta-amyloid oligomerization in the brain to prevent Alzheimer's diseaseprogression.

ALZT-OP1b (ibuprofen). The generic name is iso-butyl-propanoic-phenolicacid. ALZT-OP1b is an over the counter drug, taken in orally and doesnot require prescription. Ibuprofen has a long safety history. The drugis used for pain, fever, sports injuries and gastrointestinal problems.The weight dosage independence has been indicated on the drug package.

The investigational product ALZT-OP1b (ibuprofen) is non-selective COXinhibitor for treating inflammation as a non-steroidal anti-inflammatorydrug (NSAID). The COX enzymes convert certain fatty acids toprostaglandins. The prostaglandins at the end of the “chain” ofreactions that starts with the COX enzyme cause an increased sensitivityto pain, fever, and vasodilation (increased blood flow or inflammation).By inhibiting the start of this chain of reactions, ibuprofen thereforereduces pain, fever, and inflammation. Because ibuprofen blocks theactivity of both COX enzymes, it is considered a non-selective COXinhibitor NSAID.

As described above, dampening the neuro-inflammatory response willimpact AD progression by several mechanisms. Ibuprofen, which crossesthe human blood brain barrier (Bannworth, 1995; Parepally, 2006),dampens the production of pro-inflammatory cytokines (Gasparini, 2004),which should contribute to its utility for preventing AD progression.However, NSAIDs, such as rofecoxib and naproxen, for the treatment of ADhas been inconclusive or contributed to higher risk of AD progressionwhen administered as the sole therapy in clinical trials (Thal, 2005;Imbimbo, 2010) despite the multiple epidemiology studies showing reducedAD risk in individuals taking NSAIDs, including ibuprofen (Veld, 2001;Etminan, 2003). Besides the criticism surrounding the choice ofrofecoxib and naproxen as the NSAIDs for sole therapy in AD (Gasparini,2004), the ADAPT rofecoxib/naproxen treatment trial was conducted withsubjects exhibiting mild-to-moderate AD (Aisen 2003; Breitner, 2011).Given the epidemiology data, it has been hypothesized that NSAIDadministration may be beneficial only very early indisease (Imbimbo,2010; Breitner, 2011). The aMCI patient population is therefore thegroup that we have selected to be tested in this clinical study.

It is important to note that in the NSAID epidemiology studies, AD riskdecrease was restricted to NSAIDs that presumably lower beta-amyloid(42-)peptide levels, such as ibuprofen and indomethacin (Gasparini,2004; Imbimbo, 2010), and long-term dosing with low NSAID doses wereequally effective as higher doses (Broe, 2000; Breitner 2001). Hence, inone cohort in this AZTherapies ALZT-OP1 trial, oral ibuprofen will beadministered as tablets (ALZT-OP1b) at a dose lower (less than 5%) ofthe lowest over-the-counter approved dose. In combination with cromolynsodium inhalation treatment (ALZT-OP1a), we will test the hypothesisthat dampening the low level neuroinflammatory response with ibuprofenwill contribute significantly to preventing cognitive decline due toAlzheimer's disease progression. The dose is calculated to titrate theestimated invisible inflammatory response at the early stages of thedisease.

Uncontrolled ibuprofen dosage is associated with several side effectssuch as nausea, headache, ulcers, dizziness, and hypertension. A minornumber of cases can cause heart or renal failures. The overdose ofibuprofen can be dangerous. The proposed daily dose for this clinicaltrial is 20 fold lower than the dose over the counter, and the totalyearly dose totaled from the chronic daily dose is less than a totalweekly dose over the counter. It is not expected that the yearlytoxicity will exceed the weekly over the counter dose.

Risk Benefits of ALZT-OP1 (Cromolyn)

The main goal for using ALZT-OP1 in aMCI subject is its predictedmultifunctional treatment of the early appearance signs of cognitiveimpairment associated with Alzheimer's Disease. Low dose of ALZT-OP1a isexpected to control Aβ oligomerization and slow down the extra cellularAβ fibril brain accumulation. At the same time, low dose of ALZT-OP1acan inhibit cytokine production from the high brain must cellconcentration. The low dose ALZT-OP1b (ibuprofen), a known non-specificCOX inhibitor, is expected to control the inflammatory responseassociated with Aβ plaque formation. The main benefits of the low dosechronic daily use are to control and slow down the earlier ADpathophysiology cascade of the main events that trigger intracellulartau tangles and neuron degeneration. ALZT-OP1 treatment will slow downlater AD stages manifestation, prolong the patient's life, bettercontrol the quality of life and significantly lower the expensive costof family and nursing treatment and human resources.

Both medications are approved for treatment since the seventies. Bothdrugs displayed excellent safety profile at much higher dosages.However, each of the drugs have its own short and chronic treatment sideeffects for the used dosages.

AZLT-OP1a has a long history of safety in adults and children. Cromolynsodium is available as metered-dose inhalers, and used for long-termasthma prevention ad control by decreasing inflammation and improvinglung function. Cromolyn blocks cytokine release of mast cells that causeairways inflammation. The drug is associated with very mild sideeffects, like coughing, skin rash, and headaches. The treatment doses inthis clinical trial are 4-8 folds lower that prescribed and are notexpected to cause any significant higher toxicity that the asthma dose.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration from the specification andpractice of the invention disclosed herein. All references cited hereinfor any reason, including all journal citations and U.S./foreign patentsand patent applications, are specifically and entirely incorporatedherein by reference. It is understood that the invention is not confinedto the specific reagents, formulations, reaction conditions, etc.,herein illustrated and described, but embraces such modified formsthereof as come within the scope of the following claims.

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The invention claimed is:
 1. A method for treating neuroinflammation ina human subject in need thereof comprising co-administering: once perday by inhalation a powder comprising 1 to 100 mg of cromolyn sodium;and once per day by mouth 10 mg of ibuprofen, wherein, taken together,the cromolyn sodium and the ibuprofen are therapeutically effective. 2.The method of claim 1, wherein the powder further comprises anexcipient.
 3. The method of claim 2, wherein the excipient is lactosemonohydrate.